Dominique Lerouet

Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury

The aim of this study was to investigate the effect of N‐(3‐(aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of inducible calcium‐independent nitric oxide synthase (iNOS), on the functional and histopathological outcomes of experimental transient focal cerebral ischaemia in rats. Transient ischaemia was produced by the occlusion for 2 h of both the left middle cerebral artery and common carotid artery. Treatments with 1400W (20 mg kg−1) or vehicle were started 18 h after occlusion of the arteries and consisted in seven subcutaneous injections at 8 h interval. Ischaemic outcomes and NOS activities (constitutive and calcium‐independent NOS) were evaluated 3 days after ischaemia. 1400W significantly reduced ischaemic lesion volume by 31%, and attenuated weight loss and neurological dysfunction. 1400W attenuated the calcium‐independent NOS activity in the infarct by 36% without affecting the constitutive NOS activity. These findings suggest that iNOS activation contributes to tissue damage and that selective inhibitors of this isoform may be of interest for the treatment of stroke.

Changes in oxidative stress, iNOS activity and neutrophil infiltration in severe transient focal cerebral ischemia in rats

Oxidative stress, inducible nitric oxide synthase (iNOS) and neutrophils all contribute to post-ischemic brain damage. This study has determined the time courses of these three phenomena after ischemia in parallel with histological and functional outcomes. Ischemia was produced in rats by occluding the left middle cerebral artery and both common carotid arteries for 20 min. Regional cerebral blood flow (rCBF) rapidly decreased to 20% of its preischemic value during occlusion and stabilized at 60% following reperfusion. The striatal infarction was maximal 15 h after reperfusion (50+/-3 mm(3)), whereas the cortical infarction reached its maximum at 48 h (183+/-10 mm(3)). This drastic decrease in rCBF followed by incomplete reperfusion and massive infarction is, thus, extremely severe. The cortical infarction was strongly correlated with the neurologic deficit and loss of body weight. Oxidative stress, evaluated by the decrease in glutathione concentrations, appeared in the striatum at 6 h after reperfusion and in the cortex at 15 h. Calcium-independent NOS activity, considered as inducible NOS activity, was significantly enhanced at 24 h in the striatum and at 48 h in the cortex. Myeloperoxidase activity, a marker of neutrophil infiltration, was significantly increased at 48 h in both the striatum and cortex. These time courses show that the delayed iNOS activity and neutrophil infiltration that occur after the maturation of infarction in severe ischemia may not contribute to ischemic brain damage. By contrast, early oxidative stress may well be implicated in cerebral injury.

Antisense Oligodeoxynucleotide to Inducible Nitric Oxide Synthase Protects against Transient Focal Cerebral Ischemia—Induced Brain Injury

Nitric oxide (NO) has been suspected to mediate brain damage during ischemia. Here the authors studied the effects of an antisense oligodeoxynucleotide (ODN) directed against the inducible isoform of NO synthase (iNOS) in a model of transient focal cerebral ischemia in rats. Treatment consisted of seven intracerebroventricular injections of a phosphodiester/phosphorothioate chimera ODN (3 nmol each) at 12-hour intervals, and was initiated 12 hours before a 2-hour occlusion of the left middle cerebral artery and common carotid artery. Outcomes were measured three days after ischemia. When compared with animals treated with vehicle or an appropriate random non-sense control ODN sequence, the antisense treatment reduced the lesion volume by 30% and significantly improved recovery of sensorimotor functions, as assessed on a neuroscore. This effect was associated with a decrease in iNOS expression, as assessed by Western blot, a 39% reduction in iNOS enzymatic activity evaluated as Ca2+-independent NOS activity, and a 37% reduction in nitrotyrosine formation, reflecting protein nitration by NO-derived peroxynitrite. These findings provide new evidence that inhibition of iNOS may be of interest for the treatment of stroke.

MFGE8 inhibits inflammasome-induced IL-1β production and limits postischemic cerebral injury

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1β production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1β production by macrophages through mediation of integrin β(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1β production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1β production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1β production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1β production.

Neuroblast survival depends on mature vascular network formation after mouse stroke: role of endothelial and smooth muscle progenitor cell co-administration

Pro‐angiogenic cell‐based therapies constitute an interesting and attractive approach to enhancing post‐stroke neurogenesis and decreasing neurological deficit. However, most new stroke‐induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single‐cell‐type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood‐derived CD34+ cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co‐administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri‐infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri‐ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co‐administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke.

Induction of type 2 iodothyronine deiodinase in astrocytes after transient focal cerebral ischemia in the rat

This study investigated the expression of deiodinases of thyroid hormones in the rat brain after transient occlusion of the middle cerebral artery. The activity of type 2 deiodinase (D2), which catalyzes the deiodination of thyroxine into the more active thyroid hormone 3,5,3′-triiodothyronine, was strongly increased by cerebral ischemia at 6 and 24 hours in the striatum and at 24 hours in the cerebral cortex. The activity of type 3 deiodinase, which catalyzes the inactivation of thyroid hormones, was not affected by ischemia. In situ hybridization showed, as soon as 6 hours, an upregulation of the expression of D2 mRNA in the ipsilateral striatum, which disappeared at 24 hours. In the ipsilateral cortex, the induction of D2 mRNA started at 6 hours, was increased at 24 hours and finally declined at 72 hours. These results were confirmed by reverse transcription-PCR for D2 mRNA in the striatum and cerebral cortex. The upregulation of D2 mRNA after ischemia was mainly localized in astrocytic cell bodies. These results show that D2 is rapidly induced in astrocytes after ischemic stroke. Future work will include the exploration of the role of the upregulation of this enzyme, responsible for local 3,5,3′-triiodothyronine production as a neuroprotective mechanism in the brain.

Role of the l-arginine-nitric oxide pathway in the basal hydroxyl radical production in the striatum of awake rats as measured by brain microdialysis

In the present study, using the microdialysis technique, we provided evidence of the existence of hydroxyl radicals (.OH) in the striatum of awake rats under physiological conditions. This .OH generation was virtually abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine. On the contrary, it was significantly enhanced by the .NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The effect of L-NAME was completely reversed by L-arginine. These results suggest that the basal .OH production is largely the consequence of an NMDA receptor-mediated glutamatergic tone. Moreover, it is likely that endogenous .NO exerts an antioxidant activity in brain by preventing the rise in .OH levels.

Lack of iNOS induction in a severe model of transient focal cerebral ischemia in rats

Calcium-independent nitric oxide synthase (NOS) activity has been reported in ischemic brains and usually attributed to the inducible isoform, iNOS. Because calcium-independent mechanisms have recently been shown to regulate the constitutive calcium-dependent NOS, we proposed to confirm the presence of iNOS activity in our model of transient focal cerebral ischemia in rats. Our initial results showed that, in our model, ischemia induced an important increase in brain calcium concentration. Consequently, the determination of calcium-independent NOS activity required a higher concentration of calcium chelator than classically used in the NOS assay. In these conditions, calcium-independent NOS activity was not observed after ischemia. Moreover, our ischemia was associated with neither iNOS protein expression, measured by Western blotting, nor increased NO production, evaluated by its metabolites (nitrate/nitrite). Our results demonstrate that iNOS activity may be overestimated due to increased brain calcium concentration in ischemic conditions and also that iNOS is not systematically induced after cerebral ischemia.

Another "string to the bow" of PJ34, a potent poly(ADP-Ribose)polymerase inhibitor: an antiplatelet effect through P2Y12 antagonism?

Background Neuro- and vasoprotective effects of poly(ADP-ribose)polymerase (PARP) inhibition have been largely documented in models of cerebral ischemia, particularly with the potent PARP inhibitor PJ34. Furthermore, after ischemic stroke, physicians are faced with incomplete tissue reperfusion and reocclusion, in which platelet activation/aggregation plays a key role. Data suggest that certain PARP inhibitors could act as antiplatelet agents. In that context, the present in vitro study investigated on human blood the potential antiplatelet effect of PJ34 and two structurally different PARP inhibitors, DPQ and INO-1001. Methods and results ADP concentrations were chosen to induce a biphasic aggregation curve resulting from the successive activation of both its receptors P2Y1 and P2Y12. In these experimental conditions, PJ34 inhibited the second phase of aggregation; this effect was reduced by incremental ADP concentrations. In addition, in line with a P2Y12 pathway inhibitory effect, PJ34 inhibited the dephosphorylation of the vasodilator stimulated phosphoprotein (VASP) in a concentration-dependent manner. Besides, PJ34 had no effect on platelet aggregation induced by collagen or PAR1 activating peptide, used at concentrations inducing a strong activation independent on secreted ADP. By contrast, DPQ and INO-1001 were devoid of any effect whatever the platelet agonist used. Conclusions We showed that, in addition to its already demonstrated beneficial effects in in vivo models of cerebral ischemia, the potent PARP inhibitor PJ34 exerts in vitro an antiplatelet effect. Moreover, this is the first study to report that PJ34 could act via a competitive P2Y12 antagonism. Thus, this antiplatelet effect could improve post-stroke reperfusion and/or prevent reocclusion, which reinforces the interest of this drug for stroke treatment.